Humans have two genes that encode acetylcholine-hydrolyzing enzymes, AChE and BChE (Soreq and Zakut, 1993). The ACHE and BCHE genes, although drastically different from each other in base composition, are thought to be derived from a common ancestral gene. ACHE, mapped to chromosome 7q22 encodes the primary enzyme, acetylcholinesterase (AChE, E.C. 3.1.1.7), which terminates neurotransmission at synapses and neuromuscular junctions. BCHE, mapped to 3q26 encodes butyrylcholinesterase (BChE or alternatively BuChE, E.C. 3.1.1.8), a homologous serum esterase with somewhat broader substrate specificity. BuChE acts as a scavenger of natural and man-made poisons, including organophosphate and carbamate pesticides, that are increasingly a threat to human health (Loewenstein et al., 1993). Yet, individuals with no BuChE activity (silent phenotype) in their serum are apparently healthy.
The book Human Cholinesterases and Anticholinesterases by Soreq and Zakut (Academic Press, Inc., 1993) provides a summation of the biochemical and biological background as well as the molecular biology of human cholinesterase genes. The book in its entirety is incorporated herein by reference. Further, the book Transgenic Xenopus by Seidman and Soreq (Humana Press, 1996) provides a summation of the development of the Xenopus transgenic animal model. The book in its entirety is incorporated herein by reference.
Briefly, AChE acquires heterogeneous properties in different tumors distinct from those it displays in muscle and nerve, hemopoietic cells, embryonic tissue and germ cells. Monomers of the catalytic AChE subunit were observed in meningiomas and tetrameres in glioblastomas. Inhibition properties different from those of normal AChE were determined for serum AChE in various carcinomas. Moreover, tumorigenic expression of the corresponding ACHE gene was found to be subject to variable control mechanisms. In differentiating neuroblastoma cells, inhibition of mevallonate synthesis, which decreases proliferation rates, increases AChE levels. In PC12 cells, in contrast, nerve growth factor induces the production of hydrophilic AChE, while embryonal, carcinoma cells and thyroid tumor cells produce this enzyme under all conditions examined.
A major hydrophilic form of AChE with the potential to be "tailed" by non-catalytic subunits is expressed in brain and muscle whereas a hydrophobic, phosphoinositide (PI)-linked form of the enzyme is found in erythrocytes. Two sublines of the human erythroleukemic K-562 cells were shown to express the PI-linked form of AChE, however, with different structural properties of the PI moiety. To reveal the molecular mechanisms underlying the heterogeneous tumorigenic expression of AChE, applicants initiated the investigation of alternative splicing in AChEmRNAs from different tumor cells.
Alternative splicing controls the generation of proteins with diverse properties from single genes through the alternate excision of intronic sequences from the nuclear precursors of the relevant mRNAs (Pre-mRNA). It is known to be cell type-, tissue- and/or developmental stage-specific and is considered as the principal mechanism controlling the site(s) and timing of expression and the properties of the resultant protein products from various genes.
Alternative exons encoding the C-terminal peptide in AChE were shown to provide the molecular origins for the amphiphilic (PI)-linked and the hydrophilic "tailed" form of AChE in Torpedo electric organ. The existence of corresponding alternative exons and homologous enzyme forms in mammals suggested that a similar mechanism may provide for the molecular polymorphism of human AChE. However, the only cDNAs reported to date from mammalian brain and muscle encode the hydrophilic AChE form. Nonetheless, RNA-protection and PCR analyses have demonstrated the existence of two rare alternative AChEmRNAs in mouse hemopoietic cells.
More specifically, three alternative AChE-encoding mRNAs have been described in mammals. The dominant brain and muscle AChE found in NMJs (AChE-T) is encoded by an mRNA carrying exon E1 and the invariant coding exons E2, E3, and E4 spliced to alternative exon E6. AChEmRNA bearing exons E1-4 and alternative exon E5 encodes the glycolipid phosphatidylinositol (GPI)-linked form of AChE characteristic of vertebrate erythrocytes (AChE-H). An additional readthrough mRNA species retaining the intronic sequence I4 located immediately 3' to exon E4 was reported in rodent bone marrow and erythroleukemic cells and in various tumor cells lines of human origin (FIGS. 1A and 1B).
AChE is accumulated at neuromuscular junctions (Salpeter 1967) where it serves a vital function in modulating cholinergic neurotransmission (Reviewed by Soreq and Zakut, 1993).
Anti-cholinesterase drugs are employed to treat a variety of diseases including Alzheimer's and Parkinson's diseases, glaucoma, multiple sclerosis, and myasthenia gravis (reviewed in Millard and Broomfield). As a brief summary, glaucoma is a leading cause of blindness. Several different kinds of glaucoma exist, but the most common is primary open-angle glaucoma (POAG). Because little is known conclusively about the etiology of this disease, present medical treatment is purely symptomatic. For at least thirty years, ophthalmologists have been treating advanced POAG with anti-ChE compounds. The most often-used has been echothiophate; other agents have included DFP, neostigmine, physostigmine, paraoxon and tetraethylpyrophosphate (TEPP).
Physostigmine was first reported to mitigate the autoimmune disease, myasthenia gravis (MG), and provided the basis of a diagnostic test that enabled detection of moderate forms of the disease. This work was the impetus for uncovering the cause of organophosphorus nerve agent toxicity and, sixty years later, quaternary carbamate compounds, such as neostigmine and pyridostigmine, are still used in the symptomatic treatment of MG to provide increased neuromuscular transmission and, to some extent, greater muscular strength. Edrophonium, a reversible anti-ChE, also improves MG by compensating for reduction of ACh receptors through elevation of neurotransmitter levels.
Senile demential of Alzheimer type (SDAT) is one of the most common causes of mental debilitation among the elderly. SDAT coincides with selective degeneration of basal forebrain cortical cholinergic neurons and "neurofibrillary tangles" contain both AChE and BuChE activity. Brain AChE activity apparently is reduced in SDAT, a form of cholinergic imbalance. Several reports of specific reductions and increases in different brain AChE isoforms, as well as an abnormal SDAT-associated cerebrospinal fluid AChE isoelectric point variant have been reported. Because of the general destruction of normal presynaptic cholinergic fibers in SDAT, however, local changes in AChE may be quite distal to the cause of injury.
It has been suggested that a procedure to counter SDAT symptoms would be the inhibition of AChE to allow diffusion of ACh to become the rate limiting step of synaptic transmission and, hence, to conserve selectively the "functional" transmitter released. Thus, anti-ChEs would compensate for the diminished ACh released by the surviving cortical neurons. There was initial success in improving SDAT with arecoline and physostigmine but the latter was not sufficient to counteract completely the side-effects of inhibition. 1,2,3,4,-tetrahydro-9-aminoacridine (tacrine) has emerged as a candidate, but it is premature to conclude proof of efficacy and it is possible that it acts by stimulating ACh synthesis, as well as by inhibiting ChEs.
Furthermore, anti-cholinesterase poisons form a broad category of agricultural and household pesticides including organophosphorous and carbamate agents. There are reports of individual sensitivity to these agents and screening methods are needed to determine the safety of the agents.
Retinal photoreceptor degeneration (RD) is the most common symptom of retinitis pigmentosa, a group of inherited human blindness disorders displaying both genetic and phenotypic diversity (reviewed in Shastry, 1994). While little is known about the etiology of RD, progress in understanding this heterogenous disorder has been aided considerably by the utilization of both naturally occurring and transgenic animal strains with known distinct genomic defects, which display retinal degeneration analogous to human RD (Mullen and LaVail, 1976; Pittler and Baehr, 1991; Connell et al., 1991; Sung et al., 1994). Most of these strains carry mutated key proteins that are directly involved in the phototransduction pathway (Humphries et al., 1992). However, it is currently estimated that only 30% of the human RD patients carry mutated phototransduction proteins (Shastry, 1994). This, in turn, raises the question whether other mechanism(s) exist which explain the occurrence of RD with unknown genomic origins. Because of the known association between cholinergic imbalance and neurodeterioration (Bierer et al., 1995), applicants explored the possibility of cholinergic involvement and possible imbalance in RD.
Research and development directed toward the production of new specific, effective, low-toxicity anti-cholinesterase drugs and insecticides are abundant. However, heretofore, no effective in vivo system has been developed which would allow for the rapid, effective and reliable screening of such anti-cholinesterase substances. Applicants have developed a transgenic animal assay system as disclosed in co-pending U.S. Ser. No. 08/370,156 assigned to the same assignee and incorporated herein by reference. Additionally, the book Transgenic Xenopus by Seidman and Soreq (Humana Press, 1996) provides a summation of the development of the Xenopus transgenic animal model and Beeri et al. (1994) and Beeri et al. (1995) provide a information on the development of the transgenic mouse model.
However, in developing transgenic models carrying human genes it is useful to have the gene under the human promoter. Examples from other genes demonstrate transcriptionally important regions at distances up to 40 kb from the transcription initiation site (e.g. serum albumin). In the previous studies and in Examples herein below Applicants used the 596 bp region immediately adjacent to the transcription initiation site in the human ACHE gene (Soreq, et al., 1990; Ben Aziz-Aloya et al, 1992). As indicated in the Examples, data shows that the 596 bp partial human promoter appears to be sufficient for directing persistent CNS transcription and therefore useful in establishing transgenic animal models. However, it does not induce AChE production in other tissues (Beeri et al., 1995) or direct developmental variations in the intensity of CNS expression of ACHE. Therefore, it would be useful to isolate an additional promoter to use in the transgenic assay system.